Chromosome numbers and karyotypes of seven specific taxa of the Chilean endemic genus Placea were determined. Chromosome numbers of P. lutea, P. ornata, P grandiflora, P germainii andP. aff davidii are described for the first time. All taxa are diploid with 2«=2x=16 and karyotypes are composed of four metacentric (4 m), ten submetacentric (10 sm), and two subtelocentric (2 st) chromosomes. The most symmetrical karyotype was observed in P. lutea (AI: 6.84) while the most asymmetrical karyotype was shown by P. arzae (AI: 9.72). The constancy in karyotype formulae and high similarity in asymmetry indexes suggest that some orthoselection mechanism might be involved in Placea's chromosomal evolution. In spite that no significant karyotypic differences were observed, the species may be differentiated by their chromosomal sizes. Moreover, the tribal position of Placea and its likely relationships with other hippeastroid genera are discussed.

The aim of this work is to determine the chromosome number and the karyotype of the genus Placea, and their relationships with other Hippeastroid genera in order to increase the knowledge about the karyological diversity of this endemic genus.

MATERIALS AND METHODS

The study was conducted during March to September 2008. In order to disclose the karyotype of each Placea species germplasm from seeds and bulbs was obtained from different localities in Chile (Table I, Fig. 1). Seeds were sowed in Petri dishes with water-moistened filter paper and placed at 4°C until roots reached one-centimeter of length. Bulbs were placed in glass containers with water-moistened absorbent paper at room temperarme until roots sprouted. Root tips were pre-treated in colchicine (0.05%) for 18 h at room temperature, then fixed in a freshly prepared mixture of absolute ethanol-glacial acid acetic (3:1) for 24 h and stored in 70% ethanol. Treated root tips were hydrolyzed in HC1 IN for 10 minutes at 60°C, macerated, stained with lacto-propionic orcein and squashed on a slide (Araneda et al. 2004). Slides were made permanent using liquid nitrogen and mounted in a glycerin drop. Plates were observed using a light microscope with incorporated digital camera. Images were analyzed with Micro Measure 3.3 (Reeves 2001). The length and shape of chromosomes were determined to construct the karyotypes by using Adobe Photoshop 7.0 (Seven metaphase plates from three to ten plants per species were selected). Chromosomes were classified according to Levan et al. (1964), the abbreviations being m, sm, and st which correspond to metacentric, submetacentric, and subtelocentric chromosomes, respectively.

The following parameters were calculated in each metaphase plate for the numerical characterization of the karyotypes: mean haploid chromosome length (CL), and total complement length (TCL). These parameters were compared by one-way ANOVA and Tukey's test was carried out to test differences between each pair of means. Statistical evaluation was carried out using SPSS 14.0.

Karyotype asymmetry was estimated using intrachromosomal asymmetryindex(A1); interchromosomal asymmetry index (A2); coefficient of variation of chromosome length (CVcl); coefficient variation of centromeric index (CVci) and a new asymmetry index (AI) proposedby Paszko (2006). All the species were identified using the key provided by Traub & Moldenke (1949) and later contributions by Ravenna (1981) and Muñoz (2000). The reference materials are deposited in the herbarium of the Universidad de Concepción (CONC), Instituto Nacional de Investigaciones Agropecuarias (INIA), Jardín Botánico Nacional (JBN) and Museo Nacional de Historia Natural (SGO).

Table I. Taxa and localities of Placea used in this chromosomal study.

The analyzed parameters are summarized in Table II. Mitotic metaphases and diploid karyotypes of the species are shown in Figures 1 and 2, respectively.

All seven accessions analyzed showed a common karyotype constitutedby eight chromosome pairs of different sizes and the common karyotype formula was 4m + 10sm + 2st. In most of the taxa, the longest pair corresponded to the first metacentric pair and the shortest pair was the sixth pair (submetacentric) (Table II). The shortest chromosomes were observed in P. aff. davidii and P. lutea, while the longest chromosomes were found in P. amoena. Chromosomes of P. amoena were 30% longer than chromosomes of P. aff. davidii.

For TCL, ANOVA discriminated among taxa (F= 18.22 5, P < 0.001). Considering the CL of each chromosomal pair, all the chromosomal pairs were different among taxa (Table II). In all the assessments, with exception of the seventh and eighth chromosome pairs, P. amoena was different from each other species. In general, the examined karyotypes were asymmetrical with respect to chromosome length and heterogeneous regarding chromosome uniformity. Chromosomes with low length variability were found in P. ornata, P. aff. davidii and P. lutea (A2=0.27 to 0.28). All analyzed taxa showed high degrees of karyotype asymmetry indicatedby theirhigh A1 valúes (0.92 to 0.93). However, according to the asymmetry index (AI), the most symmetrical karyotype was observed in P. lutea (AI=6.84), while the most asymmetrical karyotype was exhibited by P. arzae (AI=9.72). The scatter diagram of CVci and CVcl (Fig. 3) showed two groups of species: one composed by the most asymmetrical karyotypes (AI=8.72-9.72) and the other with the most symmetrical karyotypes (AI=6.84-7.42). Placea amoena fell apart, with the lowest relative variation in centromere position, although still variable in chromosome length.

DISCUSSION

The chromosome number and karyotype structure, but not the total chromosome length, were the same for every Placea species. Chromosome numbers in P. aff. davidii, P. ornata, P. grandiflora and P. germainii are reported for the first time, establishing a diploid number (2«=2x=16) for the genus

The sof chromosome number in genus Placea suggests that neither polyploidy nor aneuploidy nor dysploidy have played a significant role in its diversification. This fact strongly contrast with studies that have stressed how chromosomal numeric alteration processes have played an important role in the evolution of other hippeastroid genera, such as Hippeastrum, Rhodophiala, Habranthus and Zephyranthes (Naranjo 1969, Naranjo 1974, Naranjo & Andrada 1975, Flory 1977, Naranjo & Poggio 2000).

As far as karyotypes are concerned, our results agree with previous reports for Placea amoena (4m + lOsm + 2st) (Baeza & Schrader 2004, Baeza et al. 2007b). However, the karyotype of P arzae observed in this study disagrees with the 4m + 6sm + 6st with a satellite on the short arm of chromosome 6 (st) reported by Naranjo (1985). His observations were made in specimens obtained in Cautín, Villarrica (Chile, ca. 39° S), where Placea does not occur in nature. It seems likely that the material used by Naranjo (1985) was misidentified and might correspond to a species of the related genus Rhodolirium that occurs naturally in this área. Placea and Rhodolirium share the diploid number 2«=16. However, the presence of a satellite on the short arm of the st chromosome pair has been reported in two species of the latter genus (i.e. Rhodolirium montanum Phil., cited as Phycella sp. in Palma-Rojas 2000 and cited as Rhodophiala rhodolirion (Baker) Traub in Naranjo & Poggio 2000, and Rhodolirium andícola (Poepp.) Ravenna, cited as Rhodophiala andícola (Poepp.) Traub in Naranjo & Poggio 2000). However, it would be necessary to check the herbarium voucher used by Naranjo (1985) in order to corrobórate this hypothesis.

Karyotypes of all taxa are quite similar, being mostly comprised by submetacentric chromosomes. These results suggest the existence of interspecific stability in the karyotypes of Placea. The karyotypes were considered bimodal because of the presence of two outstanding groups of different mean sizes. The total complement is occupied by two large chromosome pairs and six medium-sized pairs. This process can be explained by karyotype orthoselection, where structural chromosome mutation occurs in a certain way, or by karyotype conservation, where the lack of structural mutations preserves the chromosome morphology. In our case, the constancy of the karyotype formula and similarity in asymmetry index (AI, A1 and A2), suggest that some orthoselection mechanism might be involved (White 1973, Sanso 2002). Similar results have been found in other South American monocots, such as Rhodophiala (Naranjo & Poggio 2000), Hippeastrum (Naranjo 1969, Naranjo & Andrada 1975) and Alstroemeria (Sanso 2002).

Table II. Average length of total complement (TCL), chromosome length (CL), asymmetry índex (AI), intrachromosomal and Interchromosomal asymmetry indexes (A and A respectively) of the place's species studied.

The chromosome size is also subject to evolutionary change. Frequently, the total mass of chromosomes in a nucleus has been found to be closely related to its DNA content. Thus, a factor responsible of the range of vanation of chromosome size among species of the same genus may be polyploidy, repeatedDNAcontent, orincrease inthebasic number(Sharma & Sen 2002, Schubert 2007). The vanation of chromosome size found in this study ranges between 14.95-6.85 |am in P. amoena to 8.33-4.03 |am vcvP. aff. davidii. However, neither polyploidy nor an increase in the basic number were recorded in Placea. Therefore, differences in DNA content would be the most plausible explanation for chromosome size vanation in this genus. However, this suggestion must be confirmed with DNA content measurements (e.g. flow cytometry). In spite of the similarity inthe coefficients of vanation assessed in all the species, the scatter diagram of CVci and CVcl showed two groups of species and Placea amoena as an isolated species. This singular position of Placea amoena is correlated with both karyotype difference and its cunent taxonomic segregation. This result supports subgenus Geissea (Traub & Moldenke 1949), defined to distinguish the systematic placement of P amoena from the rest of the genus, although the present phenetic analysis has no implications on whether it conesponds to a distinct evolutionary lineage within Placea.

The systematic and phylogenetic relationships of Placea within the tribe Hippeastreae is unknown, because previous phylogenetic studies in Amaryllidaceae have included only few samples of Chilean genera and none of Placea (Meerow etal. 1999, 2000). However, Placea, Phycella, Rhodolirium and Traubia share the same basic chromosome number x=8 (Naranjo 1985, Palma-Rojas 2000, Grau & Bayer 1991, Naranjo & Poggio 2000, Baeza et al. 2009) and the presence of a capitate stigma (Ravenna 2003). These shared traits suggest that these genera might be closely related within tribe Hippeastreae.

Finally, these results should be considered as a first insight into the karyotypic evolution of Placea, however, several other aspects must be considered and explored in order to achieve a better understanding of this phenomenon. It would be very useful to carry out an extensive survey across the geographical distribution range of Placea to assess intra-and interspecific variability, and perform a phylogenetic analysis to obtain a framework of its evolutionary history.